The present invention relates to a novel protein showing an activity of glutamine:fructose-6-phosphate amidotransferase (hereinafter briefly referred to as GFAT), etc. and to a DNA coding for the protein.
In recent years, the diabetic population has increased steadily and diabetes is attracting attention as one of adult diseases. Non-insulin-dependent diabetes mellitus (NIDDM) is a type of diabetes frequently found in Japan and its early detection and timely treatment are necessary to prevent the disease. Although the factors causative of NIDDM have not been fully elucidated, recent advances have provided important new insights into this process.
It is now well established that abnormalities in insulin sensitive mechanisms and reduced secretion of insulin are causes of insufficient insulin activity in NIDDM. In Europe and America, insulin resistance is predominant in patients with NIDDM while, in Japan, insulin hyposecretion is often the major cause. With recent advances in molecular biology, the cellular and molecular mechanisms underlying insulin resistance such as the insulin receptor structure and the mechanism of signal transduction downstream of the receptor, have been investigated in detail. During the last decade, glucose transporter genes has been cloned and the relationship between mutations in the genes and the process of diabetes has been studied. However, the insulin, glucokinase and mitochondrial gene abnormalities so far elucidated, taken together, account for not more than 1% of NIDDM cases. Other gene abnormalities are to be revealed in the future.
In recent years, antidiabetic agents quite differing from the conventional oral hypoglycemic agents in the mechanism of action, such as the xcex1-glycosidase inhibitors acarbose and voglibose (Diabetes Frontier, 3, 557-564 (1992); Drugs, 46, 1025-1054 (1994); Igaku no Ayumi, 149, 591-618 (1989); Rinsho to Kenkyu (Japan. J. Clinics Exper. Med.), 67, 219-233 (1990); Rinsho to Kenkyu, 69, 919-932 (1992); Rinshoi (Clinical Medicine), 21 (supplement), 578-587 (1995)) and the insulin resistance improving agents, troglitazone and pioglitazone, (Diabetes, 37, 1549-1558 (1998); Rinsho Iyaku, 9 (supplement 3), 127-150 (1993); New Engl. J. Med., 331, 1188-1193 (1994); xe2x80x9cAtarashii Tonyobyo Chiryoyaku (New Antidiabetics)xe2x80x9d (edited by Yoshio Goto), published by Iyaku Journal Co., Osaka, (1994)) have been developed. They are expected to be soon on the market.
Meanwhile, in the United States, a biguanide derivative was approved in 1996 as an antidiabetic for general prescription (New Engl. J. Med., 333, 541-549 (1995); Diabetes Spectrum, 8, 194-197 (1995)) and is attracting attention. The above-mentioned drugs, unlike sulfonylureas (SUs) which have been used for many years in routine medical care, produce a hypoglycemic effect without promoting insulin secretion from xcex2 cells of the pancreas.
It is considered, at present, that there are nine mechanisms through which antidiabetics might be able to improve insulin resistance as follows: (1) activation of insulin receptor kinase, (2) promotion of translocation of glucose transporters, (3) correction of the action of the rate-limiting enzyme involved in glucose metabolism and correction of abnormalities in glucose metabolism, (4) inhibition of gluconeogenesis in liver, (5) promotion of glucose uptake by liver, (6) enhancement of glycogenesis in liver, (7) reduction in blood lipid level, (8) decrease in gluconeogenesis in liver as resulting from the reduction in blood lipid level, and (9) enhancement of insulin sensitivity as resulting from the reduction in blood lipid level.
GFAT is an important enzyme catalyzing the conversion of fructose-6-phosphate to glucosamine-6-phosphate, which is the rate-limiting step in the hexosamine biosynthesis pathway. Inhibitors of GFAT activity are thought to promote glucose influx by cells and thereby reducing the blood glucose level. Therefore, these inhibitors are expected to be of use as antidiabetics. Their mechanism of action is thought to be associated with the process (2) or (5) mentioned above.
While the hexosamine biosynthesis pathway metabolizes glucosamine-6-phosphate to UDP-N-acetylglucosamine, CMP-N-acetylneuraminic acid, etc., those metabolic intermediates are thought to be utilized as precursors for glycosylation of proteins or as essential substrates for the synthesis of proteoglycan and ganglioside.
Insulin activates its signal transduction pathway through binding insulin receptor and translocates glucose transporters (GLUT4 etc.) pooled within cells to the cell membrane resulting in increasing glucose influx. Glucose is metabolized by glycolysis pathway and ATP is accumulated as an energy source. When the influx of glucose is excessive, however, fructose-6-phosphate enters the hexosamine biosynthesis pathway and is converted to glucosamine-6-phosphate catalyzed by GFAT. Although detailed mechanisms remain unknown, several observations indicate that metabolites of glucosamine-6-phosphate prevent glucose transporters from translocating to cell membrane, resulting in reducing cellular glucose influx (FASEB J., 5, 3031-3036 (1991); Diabetologia, 38, 518-524 (1995); J. Biol. Chem., 266, 10115-10161 (1991): J. Biol. Chem., 266, 4706-4712 (1991); Endocrinology, 136, 2809-2816 (1995)).
Therefore, the hexosamine biosynthesis pathway is considered to control the influx of glucose by a feed-back manner. GFAT is the rate-limiting enzyme in this pathway. GFAT activity is also known to be generally high in patients with NIDDM and is considered to be one of the causes of high blood glucose levels (Diabetes, 45, 302-307 (1996)).
Hypoglycemic agents, such as inhibitors of GFAT activity, whose action is mainly directed to some other tissues than pancreas invariably improve insulin resistance in target tissues. These agents have some clinical merits in addition to their hypoglycemic activity, because of their secondary effects. When used in combination with other drugs, they are highly effective and have very bright prospects before them.
Recently a human GFAT gene has been cloned (J. Biol. Chem., 267, 25208-25212 (1992)). The gene product is a 77 kDa protein composed of 681 amino acid residues. GFAT genes have been cloned from other animal species as well. For example, a murine GFAT is highly homologous to the human GFAT (91% at the nucleotide level and 98.6% at the amino acid level), hence it is considered to be the counterpart of the human GFAT (Gene, 140, 289-290 (1994)). In addition, a yeast GFAT (J. Biol. Chem., 264, 8753-8758 (1989)) and a Escherichia coli-derived GFAT (Biochem. J., 224, 779-815 (1984)) have also been reported, each having high homology with the human GFAT. It is not known about the occurrence of a new GFAT isoform gene.
Isolation of a novel protein showing GFAT activity, if successful, would make it possible to perform further investigations to elucidate the regulatory function of GFAT in the hexosamine biosynthesis pathway and, if its expression is tissue-specific, to elucidate the tissue-specific mechanisms of glucose metabolism. A specific inhibitor of the novel GFAT protein, if developed, would make it possible to develop a hypoglycemic agent acting through novel mechanisms of action and contributing to the prevention and treatment of diabetes and diabetic complications without producing serious adverse effects.
As the result of intensive research, the inventors of the present invention succeeded in cloning a cDNA having a novel nucleotide sequence from a human brain-derived cDNA library and found that the protein encoded thereby has GFAT activity. As the result of continued investigations based on such findings, the present inventors have now completed the present invention.
The present invention provides:
(1) A protein comprising an amino acid sequence represented by SEQ ID NO:1 or a substantial equivalent thereto, or a salt thereof;
(2) The protein according to (1), which has an amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:3, or a substantial equivalent thereto;
(3) The protein according to (1) or (2), which shows an activity of glutamine:fructose-6-phosphate amidotransferase;
(4) A partial peptide of the protein according to (1), or a salt thereof;
(5) An isolated DNA comprising a DNA having a nucleotide sequence coding for the protein according to (1) or the partial peptide according to (4);
(6) The DNA according to (5), which has a nucleotide sequence represented by SEQ ID NO:4;
(7) The DNA according to (5), which has a nucleotide sequence represented by SEQ ID NO:5 or SEQ ID NO:6;
(8) A recombinant vector comprising the DNA according to (5);
(9) A transformant which is transformed with the recombinant vector according to (8);
(10) A method of producing the protein or a salt thereof according to (1), which comprises culturing the transformant according to (9) under conditions suitable to express and accumulate the protein or a salt thereof according to (1) and collect the same;
(11) A pharmaceutical composition comprising the protein according to (1), the partial peptide according to (4) or a salt thereof;
(12) The pharmaceutical composition according to (11), which is a therapeutic or prophylactic agent for hypoglycemia;
(13) A pharmaceutical composition which comprises the DNA according to (5);
(14) The pharmaceutical composition according to (13), which is a therapeutic or prophylactic agent for hypoglycemia;
(15) An antibody against the protein according to (1), the partial peptide according to (4) or a salt thereof;
(16) A method for screening for a compound, or a salt thereof, which inhibits an enzyme activity of the protein or a salt thereof according to (1), which comprises measuring and comparing the enzyme activity of the protein according to (1), the partial peptide according to (4) or a salt thereof, in cases of (i) a substrate is contacted with the protein according to (1), the partial peptide according to (4) or a salt thereof and (ii) the substrate and a test compound are contacted with the protein according to (1), the partial peptide according to (4) or a salt thereof;
(17) A kit for screening for a compound, or a salt thereof, which inhibits an enzyme activity of the protein or a salt thereof according to (1), which comprises the protein according to (1), the partial peptide according to (4) or a salt thereof;
(18) A compound, or a salt thereof, which inhibits an enzyme activity of the protein or a salt thereof according to (1), which is identified by the screening method according to (16) or the screening kit according to (17);
(19) A pharmaceutical composition which comprises a compound, or a salt thereof, which inhibits an enzyme activity of the protein or a salt thereof according to (1), which is identified by the screening method according to (16) or the screening kit according to (17);
(20) The pharmaceutical composition according to (19), which is a therapeutic or prophylactic agent for diabetes;
(21) A method for treating or preventing hypoglycemia in human or a mammal which comprises administering an effective amount of the protein according to (1), the partial peptide according to (4) or a salt thereof to human or a mammal;
(22) A method for treating or preventing hypoglycemia in human or a mammal which comprises administering an effective amount of the DNA according to (5) to human or a mammal;
(23) Use of the protein according to (1), the partial peptide according to (4) or a salt thereof for production of a therapeutic or prophylactic agent for hypoglycemia;
(24) Use of the DNA according to (5) for production of a therapeutic or prophylactic agent for hypoglycemia;
Moreover the present invention provides:
(25) A partial peptide according to (4) which has an amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or an amino acid sequence substantially equivalent thereto;
(26) A DNA comprising a DNA having a nucleotide sequence capable of hybridizing under high stringent condition with the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO: 6.
(27) A recombinant vector comprising the DNA according to (26);
(28) A transformant which is transformed with the vector according to (27);
(29) A method of producing a protein, or a salt thereof, which is encoded by the DNA according to (26), which comprises culturing the transformant according to
(28) under conditions suitable to express and accumulate the protein or a salt thereof and collect the same;
(30) A protein encoded by the DNA according to (26), or a salt thereof which is produced by the method according to (29);
(31) A method of quantitative determination of the protein according to (1), the partial peptide according to (4) or a salt thereof in a test liquid sample, which comprises
(a) competitively reacting the test liquid sample and a labeled protein according to (1), partial peptide according to (4) or a salt thereof with the antibody according to (15), and
(b) measuring the ratio of the labeled protein according to (1), partial peptide according to (4) or a salt thereof which binds to the antibody;
(32) A method of quantitative determination of the protein according to (1), the partial peptide according to (4) or a salt thereof in a test liquid sample, which comprises
(a) reacting the test liquid sample with the antibody according to (15) immobilized on an insoluble carrier and a labeled antibody according to (15) simultaneously or continuously, and
(b) measuring the activity of the labeling agent on the insoluble carrier;
(33) A pharmaceutical composition which comprises the antibody according to (15) (preferably, the antibody according to (15) which shows an activity to neutralize the activity of the protein according to (1));
(34) The pharmaceutical composition according to (33) which is a therapeutic or prophylactic agent for diabetes;
(35) An antisense DNA which has a nucleotide sequence complementary or substantially complementary to the DNA according to (5) or (26) and capable of suppressing expression of the DNA;
(36) An antisense DNA according to (35), wherein the nucleotide sequence substantially complementary to the DNA according to (5) or (26) is a nucleotide sequence having an identity of not less than about 70% (preferably not less than about 80%, more preferably not less than about 90%, most preferably not less than about 95%) to the total nucleotide sequence or part of the nucleotide sequence of the nucleotide sequence complementary to the DNA according to (5) or (26);
(37) A pharmaceutical composition which comprises the antisense DNA according to (35); and
(38) A pharmaceutical composition according to (37) which is a therapeutic or prophylactic agent for diabetes.